Discovery of New E‐Selectin Inhibitors by Virtual Screening,Fluorescence Binding Assays,and STD NMR Experiments

E‐selectin is an endothelial protein that participates in the adhesion of metastatic cancer cells, and is therefore a relevant target for antitumor therapeutic intervention. In this work, virtual screening was used to identify new E‐selectin inhibitors from a subset of drug‐like molecules retrieved from the ZINC database, including the physiological ligand sLe^x as reference structure (PDB ID: 1G1T ). Four hits were chosen and subjected to molecular dynamics simulations and fluorescence binding assays, which led to the determination of experimental dissociation constants between 333 and 1012 μm . The candidate with the highest affinity was studied by saturation transfer difference (STD) NMR experiments and complete relaxation and conformational exchange matrix analysis of saturation transfer (CORCEMA‐ST), aimed at identifying the preferable binding mode with E‐selectin. Our results revealed that this new inhibitor binds more strongly than sLe^x in the E‐selectin binding site, in good agreement with simulation predictions. These properties will prove valuable for the future design of drugs that target E‐selectin.     

A Tailor‐Made “Tag–Receptor” Affinity Pair for the Purification of Fusion Proteins

A novel affinity “tag–receptor” pair was developed as a generic platform for the purification of fusion proteins. The hexapeptide RKRKRK was selected as the affinity tag and fused to green fluorescent protein (GFP). The DNA fragments were designed, cloned in Pet‐21c expression vector and expressed in E. coli host as soluble protein. A solid‐phase combinatorial library based on the Ugi reaction was synthesized: 64 affinity ligands displaying complementary functionalities towards the designed tag. The library was screened by affinity chromatography in a 96‐well format for binding to the RKRKRK‐tagged GFP protein. Lead ligand A7C1 was selected for the purification of RKRKRK fusion proteins. The affinity pair RKRKRK‐tagged GFP with A7C1 emerged as a promising solution (K_a of 2.45×10⁵ M ^?1). The specificity of the ligand towards the tag was observed experimentally and theoretically through automated docking and molecular dynamics simulations.     

Stereospecific Effects of Oxygen‐to‐Sulfur Substitution in DNA Phosphate on Ion Pair Dynamics and Protein–DNA Affinity

Oxygen‐to‐sulfur substitutions in DNA phosphate often enhance affinity for DNA‐binding proteins. Our previous studies have suggested that this effect of sulfur substitution of both O_P1 and O_P2 atoms is due to an entropic gain associated with enhanced ion pair dynamics. In this work, we studied stereospecific effects of single sulfur substitution of either the O_P1 or O_P2 atom in DNA phosphate at the Lys57 interaction site of the Antennapedia homeodomain–DNA complex. Using crystallography, we obtained structural information on the R_P and S_P diastereomers of the phosphoromonothioate and their interaction with Lys57. Using fluorescence‐based assays, we found significant affinity enhancement upon sulfur substitution of the O_P2 atom. Using NMR spectroscopy, we found significant mobilization of the Lys57 side‐chain NH₃⁺ group upon sulfur substitution of the O_P2 atom. These data provide further mechanistic insights into the affinity enhancement by oxygen‐to‐sulfur substitution in DNA phosphate.     

Quality Control Certification of RNA Aptamer‐Based Detection

Aptamers are single‐stranded DNA or RNA molecules with a defined tertiary structure for molecular recognition. Numerous RNA aptamers with excellent binding affinity and specificity have been reported; they constitute an attractive reservoir of molecular recognition elements for biosensor development. However, RNA is relatively unstable owing to spontaneous hydrolysis and nuclease degradation. Thus, RNA aptamer‐based biosensors are prone to producing false‐positive signals. Here, we present an RNA aptamer biosensor design strategy that utilises an internal control to distinguish target binding from false‐positive signals. The sequence of a chosen RNA aptamer is expanded so that it can form three consecutive short RNA–DNA duplexes with 1) a quencher‐labelled DNA strand (Q₁DNA), 2) a dual‐fluorophore‐labelled DNA strand (F₁DNAF₂) and 3) another quencher‐labelled DNA strand (Q₂DNA). The addition of a target releases Q₂DNA from the duplex assembly, and produces the expected positive signal from F₂. However, the authenticity of target recognition is validated only if no signal is generated from F₁. We have successfully engineered two fluorescent reporters by using an RNA aptamer that binds thrombin and one that binds theophylline. Both reporters show the expected binding affinity and specificity, and are capable of reporting system malfunction when treated with nucleases and chemical denaturants. This strategy provides a simple and reliable way to ensure high‐quality detection when RNA aptamers are employed as molecular‐recognition elements.     

Drug Screening Boosted by Hyperpolarized Long‐Lived States in NMR

Transverse and longitudinal relaxation times (T_1ρ and T₁) have been widely exploited in NMR to probe the binding of ligands and putative drugs to target proteins. We have shown recently that long‐lived states (LLS) can be more sensitive to ligand binding. LLS can be excited if the ligand comprises at least two coupled spins. Herein we broaden the scope of ligand screening by LLS to arbitrary ligands by covalent attachment of a functional group, which comprises a pair of coupled protons that are isolated from neighboring magnetic nuclei. The resulting functionalized ligands have longitudinal relaxation times T₁(¹H) that are sufficiently long to allow the powerful combination of LLS with dissolution dynamic nuclear polarization (D‐DNP). Hyperpolarized weak “spy ligands” can be displaced by high‐affinity competitors. Hyperpolarized LLS allow one to decrease both protein and ligand concentrations to micromolar levels and to significantly increase sample throughput.     

NBD‐Based Green Fluorescent Ligands for Typing of Thymine‐Related SNPs by Using an Abasic Site‐Containing Probe DNA

The binding behavior of green fluorescent ligands, derivatives of 7‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD), with DNA duplexes containing an abasic (AP) site is studied by thermal denaturation and fluorescence experiments. Among NBD derivatives, N¹‐(7‐nitrobenzo[c][1,2,5]oxadiazol‐4‐yl)propane‐1,3‐diamine (NBD‐NH₂) is found to bind selectively to the thymine base opposite an AP site in a DNA duplex with a binding affinity of 1.52×10⁶ M ^?1. From molecular modeling studies, it is suggested that the NBD moiety binds to thymine at the AP site and a protonated amino group tethered to the NBD moiety interacts with the guanine base flanking the AP site. Green fluorescent NBD‐NH₂ is successfully applied for simultaneous G>T genotyping of PCR amplification products in a single cuvette in combination with a blue fluorescent ligand, 2‐amino‐6,7‐dimethyl‐4‐hydroxypteridine (diMe‐pteridine).     

Novel Antitumor L‐Arabinose Derivative of Indolocarbazole with High Affinity to DNA

Novel indolocarbazole derivative 12‐(α‐L ‐arabinopyranosyl)indolo[2,3‐α]pyrrolo[3,4‐c]carbazole‐5,7‐dione (AIC) demonstrated high potency (at submicromolar concentrations) against the NCI panel of human tumor cell lines and transplanted tumors in vivo. In search of tentative targets for AIC, we found that the drug formed high affinity intercalative complexes with d(AT)₂₀, d(GC)₂₀ and calf thymus DNA (binding constants (1.6×10⁶) M ^?1≤K_a≤(3.3×10⁶) M ^?1). The drug intercalated preferentially into GC pairs of the duplex. Importantly, the concentrations at which AIC formed the intercalative complexes with DNA (C≤1 μM ) were identical to the concentrations that triggered p53‐dependent gene reporter transactivation, the replication block, the inhibition of topoisomerase I‐mediated DNA relaxation and death of HCT116 human colon carcinoma cells. We conclude that the formation of high affinity intercalative complexes with DNA is an important factor for anticancer efficacy of AIC.     

Towards Keratan Sulfate – Chemoenzymatic Cascade Synthesis of Sulfated N‐Acetyllactosamine (LacNAc) Glycan Oligomers

We report on a novel chemoenzymatic cascade for the synthesis of sulfated N‐acetyllactosamine [(3Galβ1,4GlcNAcβ1,)_n, LacNAc] oligomer structures. Starting from a linker modified GlcNAc substrate di‐ and trisaccharides were first synthesized by sequential use of human β4‐galactosyltransferase‐1 (β4GalT‐1) and β3‐N‐acetylglucosaminyltransferase from Helicobacter pylori (β3GlcNAcT). Subsequent regioselective chemical sulfation rendered the C‐6 mono‐, di‐, and tri‐O‐sulfated products in good yields. Further enzymatic elongation by β4GalT‐1 and β3GlcNAcT in a sequential mode yielded 6‐O‐sulfated LacNAc oligomers up to hexasaccharide length with variable degrees of sulfation. These carbohydrate structures mimic the sulfation pattern found in keratan sulfate and are potential ligands for different classes of glycan binding proteins.

     

Stereocontrolled Synthesis and Pharmacological Evaluation of Azetidine‐2,3‐Dicarboxylic Acids at NMDA Receptors

The four stereoisomers of azetidine‐2,3‐dicaroxylic acid (L ‐trans‐ADC, L ‐cis‐ADC, D ‐trans‐ADC, and D ‐cis‐ADC) were synthesized in a stereocontrolled fashion following two distinct strategies: one providing the two cis‐ADC enantiomers and one giving access to the two trans‐ADC enantiomers. The four azetidinic amino acids were characterized in a radioligand binding assay ([³H]CGP39653) at native NMDA receptors: L ‐trans‐ADC showed the highest affinity (K_i=10 μM ) followed by the D ‐cis‐ADC stereoisomer (21 μM ). In contrast, the two analogues L ‐cis‐ADC and D ‐trans‐ADC were low‐affinity ligands (>100 and 90 μM , respectively). Electrophysiological characterization of the ADC compounds at the four NMDA receptor subtypes NR1/NR2A, NR1/NR2B, NR1/NR2C, and NR1/NR2D expressed in Xenopus oocytes showed that L ‐trans‐ADC displayed the highest agonist potency at NR1/NR2D (EC₅₀=50 μM ), which was 9.4‐, 3.4‐, and 1.9‐fold higher than the respective potencies at NR1/NR2A–C. D ‐cis‐ADC was shown to be a partial agonist at NR1/NR2C and NR1/NR2D with medium‐range micromolar potencies (EC₅₀=720 and 230 μM , respectively). A subsequent in silico ligand–protein docking study suggested an unusual binding mode for these amino acids in the agonist binding site.     

Structure–Activity Relationships of Quinoxaline‐Based 5‐HT3A and 5‐HT3AB Receptor‐Selective Ligands

Until recently, discriminating between homomeric 5‐HT₃A and heteromeric 5‐HT₃AB receptors was only possible with ligands that bind in the receptor pore. This study describes the first series of ligands that can discriminate between these receptor types at the level of the orthosteric binding site. During a recent fragment screen, 2‐chloro‐3‐(4‐methylpiperazin‐1‐yl)quinoxaline (VUF10166) was identified as a ligand that displays an 83‐fold difference in [³H]granisetron binding affinity between 5‐HT₃A and 5‐HT₃AB receptors. Fragment hit exploration, initiated from VUF10166 and 3‐(4‐methylpiperazin‐1‐yl)quinoxalin‐2‐ol, resulted in a series of compounds with higher affinity at either 5‐HT₃A or 5‐HT₃AB receptors. These ligands reveal that a single atom is sufficient to change the selectivity profile of a compound. At the extremes of the new compounds were 2‐amino‐3‐(4‐methylpiperazin‐1‐yl)quinoxaline, which showed 11‐fold selectivity for the 5‐HT₃A receptor, and 2‐(4‐methylpiperazin‐1‐yl)quinoxaline, which showed an 8.3‐fold selectivity for the 5‐HT₃AB receptor. These compounds represent novel molecular tools for studying 5‐HT₃ receptor subtypes and could help elucidate their physiological roles.     

2-Aminopurine Fluorescence Spectroscopy for Probing a Glucose Binding Aptamer

Glucose is the most important analyte for biosensors. Recently a DNA aptamer was reported allowing binding-based detection. However, due to a relatively weak binding affinity, it is difficult to perform binding assays to understand the property of this aptamer. In this work, we replaced the only adenine base in the aptamer binding pocket with a 2-aminopurine (2AP) and used fluorescence spectroscopy to study glucose binding. In the selection buffer, glucose increased the 2AP fluorescence with a K_d of 15.0 mM glucose, which was comparable with the 10 mM K_d previously reported using the strand displacement assay. The binding required two Na⁺ ions or one Mg²⁺ that cannot be replaced by Li⁺ or K⁺. The binding was weaker at higher temperature and its van't Hoff plot indicated enthalpy-driven binding. While other monosaccharides failed to achieve saturated binding even at high concentrations, two glucose-containing disaccharides, namely trehalose and sucrose, reached a similar fluorescence level as glucose although with over 10-fold higher K_d values. Detection limits in both the selection buffer (0.9 mM) and in artificial interstitial fluids (6.0 mM) were measured.     

Cell‐Selective,Apoptosis‐inducing Rhodium(III) Crown Thiaether Complexes

Half‐sandwich rhodium(III) polypyridyl (pp) complexes with the metal atom capped by the facial crown thiaether 1,4,7‐trithiacyclononane [9]aneS₃ represent a promising class of apoptosis‐inducing potent cytostatic agents. The necrotic damage caused by the complexes is negligible. In vitro cytotoxicity assays with the human cancer cell lines MCF‐7 and HT‐29 and immortalized HEK‐293 cells indicate that the dicationic κ²N(imino) complexes [([9]aneS₃)RhCl(pp)]²⁺ are much more active than monocationic complexes [([9]aneS₃)RhCl₂(L)]⁺ (L=imidazole, CH₃CN). Whereas the κ²N(amino) complex [([9]aneS₃)RhCl(piperazine)]²⁺ is inactive, replacing piperazine with the structurally analogous κ²S (thiaether) ligand 1,4‐dithiane restores cytotoxicity as evidenced by IC₅₀ values in the range 8.1‐11.6 μM . Spectroscopic (CD, UV/Vis, NOESY) and viscosity measurements indicate that the active dppz complex 8 (IC₅₀ values: 4.7–8.9 μM ) exhibits strong intercalative binding towards DNA whereas the even more potent bipyrimidine complex 9 (IC₅₀ values: 0.6–1.9 μM ) causes no alteration of the duplex B conformation. Weaker intercalative binding is observed for the dpq complex 7 . A comparative annexin V–propidium iodide binding assay with lymphoma (BJAB) cells and healthy leukocytes demonstrates that the cytotoxic activity of complex 8 and particularly complex 9 is highly selective towards the malignant cells.     

A Single‐Electrode,Dual‐Potential Ferrocene–PNA Biosensor for the Detection of DNA

A Fc–PNA biosensor (Fc: ferrocenyl, C₁₀H₉Fe) was designed by using two electrochemically distinguishable recognition elements with different molecular information at a single electrode. Two Fc–PNA capture probes were therefore synthesized by N‐terminal labeling different dodecamer PNA sequences with different ferrocene derivatives by click chemistry. Each of the two strands was thereby tethered with one specific ferrocene derivative. The two capture probes revealed quasi‐reversible redox processes of the Fc^0/+ redox couple with a significant difference in their electrochemical half‐wave potentials of ΔE_1/2=160 mV. A carefully designed biosensor interface, consisting of a ternary self‐assembled monolayer (SAM) of the two C‐terminal cysteine‐tethered Fc–PNA capture probes and 6‐mercaptohexanol, was electrochemically investigated by square wave (SWV) and cyclic voltammetry (CV). The biosensor properties of this interface were analyzed by studying the interaction with DNA sequences that were complementary to either of the two capture probes by SWV. Based on distinct changes in both peak current and potential, a parallel identification of these two DNA sequences was successful with one interface design. Moreover, the primary electrochemical response could be converted by a simple mathematical analysis into a clear‐cut electrochemical signal about the hybridization event. The discrimination of single‐nucleotide polymorphism (SNP) was proven with a chosen single‐mismatch DNA sequence. Furthermore, experiments with crude bacterial RNA confirm the principal suitability of this dual‐potential sensor under real‐life conditions.     

A Glutathione‐Responsive Short Sequence of Metal–Organic Complex Array

A short metal–organic complex array (MOCA) containing a sequence of RPtRRu ( 1_Cl ) was found to exhibit unique responses to a major biothiol, glutathione (GSH). Upon binding of GSH to 1_Cl , the resultant 1:1 complex ( 1_GS ) formed nanofibrous assemblies that suggested supramolecular polymerization through the double‐salt‐bridge structure formation. The binding behavior of this MOCA sequence to calf thymus DNA was also dependent on GSH; a larger conformational change of DNA was observed upon binding with 1_GS , relative to that with 1_Cl .     

Ultrasensitive DNAzyme‐Based Ca2+ Detection Boosted by Ethanol and a Solvent‐Compatible Scaffold for Aptazyme Design

Functional DNA includes aptamers and DNAzymes, and metal ions are often important for achieving the chemical functions of such DNA. Biosensors based on functional DNA have mainly been tested in aqueous buffers. By introducing organic solvents with much lower dielectric constants, the interaction between metal ions and DNA can be significantly enhanced, and this might affect the performance of DNA‐based biosensors. In this work, the effect of ethanol on the activity of the EtNa DNAzyme was studied for Ca²⁺ detection. With 30 % ethanol, the sensor has a detection limit of 1.4 μm Ca²⁺, which is a 16‐fold improvement relative to that in water. This EtNa DNAzyme is unique because other tested DNAzymes are all inhibited by 50 % ethanol. Finally, by using the EtNa DNAzyme as a scaffold, the adenosine monophosphate (AMP) aptamer was inserted to construct an aptazyme, which allowed the measurement of AMP in ethanol. In summary, this study has reported the most sensitive DNA‐based sensor for Ca²⁺, and its sensitivity and selectivity can approach those of proteins or small‐molecule ligands. This work also provides a way to measure aptamer binding in organic solvents.     

Arene–RuII Complexes of Curcumin Exert Antitumor Activity via Proteasome Inhibition and Apoptosis Induction

Organometallic ruthenium(II) complexes of general formula [(η⁶‐arene)Ru(curcuminato)Cl], with arene being p‐iPrC₆H₄Me ( 1 ), C₆H₆ ( 2 ), and C₆Me₆ ( 3 ), were synthesized, characterized, and evaluated for their antitumor effects. Specifically, we explored their ability to regulate the proteasome, a validated pharmacological target in cancer treatment. Ruthenium complexes inhibited isolated proteasomes to various extents, with the biological activity of these complexes depending on the nature of the bound arene; in particular, [(η⁶‐arene)Ru(curcuminato)Cl] 2 suppressed proteasomal activities more potently than 1 , 3 , or free curcumin. Each complex also inhibited proteasomes in cultured colon cancer cells and consequently triggered apoptosis, with the [(η⁶‐benzene)Ru(curcuminato)Cl] complex 2 being the most active. The influence on the oxidative status of HCT116 cells and the DNA binding ability of the [(η⁶‐arene)Ru(curcuminato)Cl] complexes were studied. Complex 2 showed the highest antioxidant capacity; moreover, complexes 1 and 2 were shown to bind isolated DNA with higher affinity (up to threefold) than free curcumin. Collectively, our results demonstrate that the complexation of curcumin with ruthenium(II) is a promising starting point for the development of curcumin‐based anticancer drugs.     

A Virtual Screening Study of the 18 kDa Translocator Protein using Pharmacophore Models Combined with 3D‐QSAR Studies

In the present study, we considered various pharmacophore hypotheses for TSPO ligands and an optimal one was selected on the basis of 3D‐QSAR studies. This hypothesis was used in a ligand‐based virtual screening study on the Maybridge database with the aim of identifying new TSPO ligands. Binding assays revealed that all selected compounds displayed TSPO affinity at 10 μM , and among them two compounds exhibited sub‐micromolar K_i values. These results validated our applied methodologies, and the two compounds with sub‐micromolar affinity could be used as interesting leads for the development of new active TSPO ligands.     

N‐(Anilinoethyl)amides: Design and Synthesis of Metabolically Stable,Selective Melatonin Receptor Ligands

The class of N‐(anilinoethyl)amides includes melatonin receptor ligands with varied subtype selectivity and intrinsic activity. One of these ligands, the MT₂‐selective partial agonist UCM765 (N‐{2‐[(3‐methoxyphenyl)phenylamino]ethyl}acetamide), had evidenced hypnotic effects in rodents at doses ≥40 mg kg^?1 (s.c.), in spite of its sub‐nanomolar affinity for human melatonin receptors. Supposing that its low in vivo potency could be due, at least in part, to metabolic liability in rat liver, UCM765 was incubated with rat liver S9 fraction and rat, mouse, or human microsomes, and the major metabolites were identified by LC–MS, synthesized, and in vitro tested for their affinity toward MT₁ and MT₂ receptors. The obtained information was exploited to design novel analogues of UCM765 that are more resistant to in vitro oxidative degradation, while maintaining a similar binding profile. The analogue UCM924 (N‐{2‐[(3‐bromophenyl)‐(4‐fluorophenyl)amino]ethyl}acetamide) displayed a binding profile similar to that of UCM765 on cloned human receptors (MT₂‐selective partial agonist) and a significantly longer half‐life in the presence of rat liver S9 fraction.     

Impact of the Proline Residue on Ligand Binding of Neurotensin Receptor 2 (NTS2)‐Selective Peptide–Peptoid Hybrids

To investigate the binding mode and structure–activity relationships (SARs) of selective neurotensin receptor 2 (NTS2) ligands, novel peptide–peptoid hybrids that simulate the function of the endogenous ligand were developed. Starting from our recently described NTS2 ligands of type 1 , structural variants of type 2 and the metabolically stable analogues 3 a , b were developed. Replacement of the proline unit by a collection of structural surrogates and evaluation of the respective molecular probes for NTS2 affinity and selectivity indicated similar SARs as described for NT(8–13) derivatives bound to the subtype NTS1. Peptide–peptoid hybrids 2 d , 3 a , b showed substantial NTS2 binding affinity (K_i=8.1–16 nM ) and 2400–8600‐fold selectivity over NTS1. The thiazolidine derivative 3 b showed metabolic stability over 32 h in a serum degradation assay. In an inositol phosphate accumulation assay, the neurotensin mimetics 3 a and 3 b displayed an inhibition of constitutive activity exceeding 1.7–2.0 times the activity of NT(8–13). The fluorinated derivative 3 a could afford attractive opportunities to detect NTS2 by ¹⁹F magnetic resonance imaging.     

Salt-Toggled Capture Selection of Uric Acid Binding Aptamers

Uric acid is the end-product of purine metabolism in humans and an important biomarker for many diseases. To achieve the detection of uric acid without using enzymes, we previously selected a DNA aptamer for uric acid with a K_d of 1 μM but the aptamer required multiple Na⁺ ions for binding. Saturated binding was achieved with around 700 mM Na⁺ and the binding at the physiological condition was much weaker. In this work, a new selection was performed by alternating Mg²⁺-containing buffers with Na⁺ and Li⁺. After 13 rounds of selection, a new aptamer sequence named UA-Mg-1 was obtained. Isothermal titration calorimetry confirmed aptamer binding in both selection buffers, and the K_d was around 8 μM. The binding of UA-Mg-1 to UA required only Mg²⁺. This is an indicator of successful switching of metal dependency via the salt-toggled selection method. The UA-Mg-1 aptamer was engineered into a fluorescent biosensor based on the strand-displacement assay with a limit of detection of 0.5 μM uric acid in the selection buffer. Finally, comparison with the previously reported Na⁺-dependent aptamer and a xanthine/uric acid riboswitch was also made.